Spinal interneuron development is altered in Onecut constitutive mutants and in embryos depleted in spinal motor neurons.

(A-C) In constitutive Oc1/Oc2 double-mutant embryos, which additionally lack OC-3 in the spinal cord, a subset of Foxp2+ V1 INs containing the MafA transcriptional regulator (yellow) increased in cell number and expanded in an abnormal ventro-medial location as compared to control embryos at embryonic day (e)11.5 (arrows; control left hemicord, mutant right hemicord). Similarly, V1 INs abnormally producing Prdm8 and Otp (cyan) were observed in the same ventro-medial position at e12.5 (arrow). (D-J) After genetic ablation of ∼90% of spinal MNs, the number of V0v INs characterized by the presence of Evx1, and of V1 INs assessed by the production of Foxd3, was significantly reduced at thoracic levels of the spinal cord. In contrast, the number of V2b INs, which contain Gata3, was increased at brachial levels, whereas V2a INs identified by the presence of Chx10 were unchanged. Scale bars=100μm. n=3; *=adjusted p value < 0.05; ***=adjusted p value < 0.001

Spinal interneuron number and positioning is altered when Onecut factors are absent from the spinal motor neurons.

(A-L) In MN conditional Oc1/Oc2 double-mutant embryos (cDKO) at e12.5, the number of V2c INs identified by the presence of Sox1 outside of the ventricular zone was significantly reduced at all levels of the spinal cord. Furthermore, the positioning of the V1 (Foxp2), V2a (Chx10), V2b (Gata3) and V2c INs on the transversal plane of the spinal cord was significantly altered. Scale bars=100μm. ML: medio-lateral axis; DV: dorso-ventral axis. n=3; *=adjusted p value < 0.05; **=adjusted p value < 0.01; ***=adjusted p value < 0.001

Ntf3 is expressed in spinal motor neurons under the control of the Onecut transcription factors.

(A) RNA-sequencing comparison of the transcriptome of control and of OC-deficient MNs at e10.5. The spinal cord of control or MN conditional Oc1/Oc2 double-mutant embryos was isolated, dissociated, YFP-positive MNs were FACS-sorted, RNA was extracted and compared by bulk RNA sequencing. (B) Transcriptomic analysis unveiled changes in the expression levels of selected genes. MN-specific expression and changes in expression levels were confirmed by in situ hybridization (n=5). (C) Expression of the neurotrophic factor Ntf3 was specific to MNs and increased in the absence of Onecut factors (green arrows). (D) TrkC, the cognate receptor of Ntf3, was broadly detected in dorsal root ganglia (DRG) and in the ventral half of the spinal cord except in the motor columns (green arrow). Scale bars=100μm.

Spinal interneuron number and positioning is altered when Ntf3 is inactivated in the spinal motor neurons.

(A-L) In MN conditional Ntf3 mutant embryos (cNtf3) at e12.5, the number of V1 (Foxp2) and of V2a (Chx10) INs was significantly increased at all levels of the spinal cord, while V2b (Gata3) only expanded at thoracic level, and V2c (Sox1, white arrows) were not altered. Furthermore, the positioning of all these IN populations on the transversal plane of the spinal cord was altered at all levels of the spinal cord. Scale bars=100μm. ML: medio-lateral axis; DV: dorso-ventral axis. n=3; *=adjusted p value < 0.05; **=adjusted p value < 0.01; ***=adjusted p value < 0.001

Locomotor coordination is altered when Ntf3 is inactivated in the spinal motor neurons.

(A-C) In female mice, while control animals demonstrated proper motor coordination and efficient motor learning evidenced by the increase in rotarod latency with time, animals carrying a conditional Ntf3 mutation in MNs (cNtf3) were almost unable to cope with rotarod acceleration and initially showed increased balance beam latency and footslips, which however normalized with time. (D-F) In male mice, conditional Ntf3 mutants showed motor coordination and learning defects on the rotarod but didn’t demonstrate any alteration in the balance beam test. n=8 in each group; *= p value < 0.05; **= p value < 0.01; ***= p value < 0.001

Spinal interneuron number and positioning is altered when Onecut factors are absent from the spinal motor neurons.

(A-H) In MN conditional Oc1/Oc2 double-mutant embryos (cDKO) at e14.5, the number of V2c INs identified by the presence of Sox1 outside of the ventricular zone was significantly reduced at all levels of the spinal cord. Furthermore, the positioning of the V1 (Foxp2), V2a (Chx10), V2b (Gata3) and V2c INs on the transversal plane of the spinal cord was altered. ML: medio-lateral axis; DV: dorso-ventral axis. n=3; *=adjusted p value < 0.05; **=adjusted p value < 0.01; ***=adjusted p value < 0.001.

Ntf3 overexpression after chicken embryonic spinal cord electroporation mildly impacts spinal interneuron development.

(A) The pHb9-Ntf3-IRES-EGFP expression vector was electroporated in the chicken embryonic spinal cord. (B) Immunofluorescence labelings for Isl1 to identify MNs (given its transient expression, maximal GFP production could only be visualized 24h after electroporation) and Chx10 to visualize V2a INs 48h after electroporation. (C) Quantification of IN populations in control (Ctrl, contralateral sides) or electroporated (E) sides of the spinal cord. The number of V2a INs was mildly decreased after Ntf3 overexpression, while other IN populations were not affected. Scale bars=100μm. n=3; *=adjusted p value < 0.05.

Spinal interneuron number and positioning is altered when Ntf3 is inactivated in the spinal motor neurons.

(A-H) In MN conditional Ntf3 mutant embryos (cNtf3) at e14.5, the number of V1 (Foxp2) INs was significantly increased at all levels of the spinal cord, while V2a (Chx10) only expanded at lumbar level and V2b (Gata3) or V2c (Sox1, white arrows) were not altered. Furthermore, the positioning of all these IN populations on the transversal plane of the spinal cord was altered at all levels of the spinal cord. Scale bars=100μm. ML: medio-lateral axis; DV: dorso-ventral axis. n=3; *=adjusted p value < 0.05; **=adjusted p value < 0.01; ***=adjusted p value < 0.001.